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Journal of Enzyme Inhibition and Medicinal Chemistry
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Amidine Derivatives and Leishmania amazonensis:
an Evaluation of the Effect of Nitric Oxide (NO) Production on the Parasite-macrophage Interaction
R.M. Temporal, L. Cysne-Finkelstein, A. Echevarria, A.J. Silva-Gonçalves, L.L.
Leon & M.S. Genestra
To cite this article: R.M. Temporal, L. Cysne-Finkelstein, A. Echevarria, A.J. Silva-Gonçalves, L.L.
Leon & M.S. Genestra (2005) Amidine Derivatives and Leishmania�amazonensis: an Evaluation of the Effect of Nitric Oxide (NO) Production on the Parasite-macrophage Interaction, Journal of Enzyme Inhibition and Medicinal Chemistry, 20:1, 13-18, DOI: 10.1080/14756360400015207 To link to this article: https://doi.org/10.1080/14756360400015207
Published online: 03 Oct 2008.
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Amidine Derivatives and Leishmania amazonensis: an Evaluation of the Effect of Nitric Oxide (NO) Production on the Parasite-macrophage Interaction
R.M. TEMPORAL1, L. CYSNE-FINKELSTEIN1, A. ECHEVARRIA2, A.J. SILVA-GONC¸ ALVES3, L.L. LEON1, & M.S. GENESTRA1
1Department of Immunology, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, Brazil,2Department of Chemistry, Universidade Federal Rural do, Rio de Janeiro, Brazil, and3Department of Biochemistry and Molecular Biology, Instituto Oswaldo Cruz /FIOCRUZ, Rio de Janeiro, Brazil
(Received 13 February 2004; In final form 15 June 2004)
Abstract
Previous work has demonstrated that N-N0-diphenyl-R-benzamidine was highly effective against Leishmania amazonensis promastigotes/axenic amastigotes andTrypanosoma evansitrypomastigotes and the compound with a methoxy substituent, was the most effective derivative in the parasite-macrophage interaction. Comparative analysis of the nitric oxide (NO) released from the culture infection’s supernatant showed the amidine to be less effective than pentamidine Isethionate as a reference drug. Additionally, in order to verify if the methoxylated derivative interferes with NO production byL. amazonensis, the effect of the amidine on the constitutive nitric oxide synthase (cNOS) purified from parasites, was examined, but demonstrated less activity in comparison with the reference drug. This data contributes to studies concerning the metabolic targets present inLeishmaniaparasites for leishmanicidal drugs.
Keywords: Leishmania amazonensis, nitric oxide, Nitric oxide synthase, pentamidine, methoxy-amidine
Introduction
Nitric oxide (NO) is a small radical biosynthesized from L-arginine by the enzyme nitric oxide synthase (NOS) in a two stage oxidation reaction that results in two products:L-citruline and NO, the latter with an extremely short life [4,10]. A NOS family exists in mammals, that includes a constitutive isoform (cNOS) expressed in certain neuronal (nNOS) and endothelial (eNOS) cells, in contrast with the inducible isoform (iNOS), expressed preferably in macrophages [33]. All the NOS enzymes so for characterized are hemoprotein dimers comprised of subunits and required cofactors such as NADPH, tetrahydrobiopterin (H4B), FAD and FMN. All NOS isoforms are regulated by calmodulin, while nNOS and eNOS regulation depends on Ca2þ concentrations. NO is a labile
molecule, and it may play important biological functions in the cell in which it is synthesized, and in its interaction with molecules within neighboring cells.
The lipophilic properties of NO allows an easy diffusion through the membranes and on the cellular environment, but this radical can be stabilized or degraded by its interaction with diverse intra- and extracellular molecular residues [28].
Leishmania parasites are associated with a wide spectrum of clinical forms of the disease, from cutaneous infections to severe visceral or diffuse cutaneous leishmaniasis, turning into a major public health problem. The control of the disease in the New World is difficult due to the great variety of different species and its clinical manifestations. The drugs chosen for treatment of all clinical forms of leishmaniasis are sodium stibogluconate (Pentostam)
ISSN 1475-6366 print/ISSN 1475-6374 onlineq2005 Taylor & Francis Ltd DOI: 10.1080/14756360400015207
Correspondence: R. M. Temporal, Department of Immunology, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, Brazil. Fax: 5 21 2280 1589. E-mail: temporal@ioc.fiocruz.br
and meglumine antimonate (Glucantime). Alternative drugs, such as pentamidine, amphotericin B and some azo-derivatives, are highly toxic, producing serious side effects. NO production appears to constitute one of the main microbiocidal mechanisms of the murine macrophage [27] and has been implicated in the elimination of several microorganisms, including Leishmania.
The mechanisms of action of NO onLeishmaniaare not well established, but it may act together with reactive oxygen species (ROS) in order to damage microbial DNA, proteins and lipids. NO can covalently react with intracellular iron, thus reacting with Fe – S prosthetic groups of susceptible enzymes (e.g. aconi- tase, complex I and II of the mitochondrial electron transport chain) resulting in the formation of iron- nitrosyl complexes with inactivation and degradation of these enzymes and cessation of replication. It has been confirmed by several laboratories that control of Leishmania infection in the murine model is NO- dependent [9,29].
Moreover, recently it has been reported that L. amazonensis also produces NO (detected as nitrite in the culture supernatant); it can be hypothesized that, depending of NO concentration, there exists a cross-talk or a down regulation mechanism involving parasites and host cells, favorable or not to the establishment of infection [1,16 – 18]. Inherently to these aspects, NO participation in other host-parasites interactions has been described [24,31] so that, the NO pathway of Leishmania parasites can constitute a good target for leishmanicidal drugs, without interference with NO production by the host cell. This work aims to show NO production during the host cell-parasite interaction and the interference of certain drugs against the defense systems ofL. amazonensis.
Materials and methods
Reagents
Benzamidine, trypsin inhibitor, penicillin G, KCl, leupeptin, L-glutamine, Schneider’s Insect Medium, MgCl2, phenylmethylsulfonyl fluoride (PMSF), N-1-naphthylethylenediamine, phosphoric acid, sulfa- nilamide, sucrose, Tris HCl, dithiotritol (DTT), aprotinin, L-arginine, NADH, NADP, NADPH, EDTA, (6R)-5,6,7,8-tetrahidrobiopterin (H4B), 20-50- ADP agarose were from Sigma Chemical Co., St. Louis, MO (USA). Glycerol was from Bio Rad (USA).
Fetal calf serum (FCS) was from Gibco BRL (USA).
Amicon was obtained from Danvers, MA (USA).
Parasites
Leishmania amazonensis promastigotes (MHOM/BR/
77/LTB0016 strain) were grown at 268C in Schnei- der’s medium supplemented with 10% (v/v) of fetal calf serum (FCS), pH 7.2 at the late log phase of
growth [11]. Parasites were harvested from the culture medium (with/without the drugs), counted in a Neubauer’s chamber and adjusted to a concentration of 4 £ 106promastigotes/mL.
Synthesis
N,N0-diphenyl-4-OCH3-benzamidine was synthe- sized from the corresponding 4-OCH3-benzanilide with phosphorus pentachloride (PCl5), giving 4- OCH3-benzimidoyl chlorides in situ, followed by the addition of aniline to afford the target compounds.
The amidine derivative in 80– 90%, yield was recrys- tallized from methanol/toluene and fully characterized by IR,1H and13C NMR and mass spectrometry [12].
Drug assay
For the “in vitro” assay, experiments were carried out in triplicate as follows: 1) Balb/c mice peritoneal macrophages were cultured in presence/absence of methoxy-amidine; 2) parasites (not treated/treated 24 h-268C with the drugs) were added in a proportion of parasite:macrophage of 5:1; 3) drugs were added after infection was installed and 4) a control without parasite. The drug concentrations added in all assays corresponded to the LDs50 of the compounds. All tests were done using Pentamidine Isethionate (Figure 1) as the reference drug [34].
Nitrite determination
Macrophage culture supernatants were used to test NO production. Nitrite, a stable breakdown of NO, was measured spectrophotometrically by adding Griess reagent (0.1% N-1-naphthylethylenediamine in 5% phosphoric acid and 1% sulfanilamide) to the same volume of culture supernatant. After 10 – 15 min at room temperature, the absorbance of the mixture was measured at 540 nm. Nitrite concentrations were estimated by comparison with a standard curve prepared with sodium nitrite [21].
Purification of NOS from L. amazonensis promastigotes A cell-free extract was prepared from promastigotes (cultures containing ,73% of metacyclic forms) by freeze-thawing the cell suspension (5 £ 109cells mL21) 3–5 times and sonicating for 5 £ 45 s over ice in 0.25 mol/L sucrose containing 5 mmol/L KCl. The contents were centrifuged at 10,000g for 30 min and the supernatant was adjusted with antiproteolytic buffer containing: 0.1 mmol/L PMSF (phenylmethylsulfonyl fluoride), 0.01% (p/v) leupeptin, 0.2 mg/mL trypsin inhibitor and 1 mmol/L benzamidine to a final volume of 5 mL. The crude soluble extract was centrifuged at 100,000g for 60 min at 48C and the supernatant (1.01 mg/mL protein) was applied to a column R.M. Temporalet al.
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(8.5 £ 1.5 cm) packet with 20,50- ADP agarose equilibrated with 10 mmol/L Tris–HCl, pH 7.5 contain- ing 1 mmol/L DTT (dithiotreitol), 1 mmol/L EDTA, 0.5 mmol/L PMSF, 25 units/ml aprotinin and 0.5 mmol/L L-arginine (buffer A). The column was then washed successively with 20 mL of buffer A, 20 mL of buffer A containing 0.5 mmol/L NADH, 20 mL of buffer A contaning 0.5 mmol/L NADP and 20 mL of buffer A. The enzyme was eluted with 10 mL of buffer A containing 10 mmol/L NADPH, 3mmol/L H4B and 10% (v/v) glycerol and concentrated to 2 mL using an Amicon concentrator (Danvers, MA, USA). The protein concentration (126mg/ml) was estimated spectrophotometrically (260–280 nm) and the samples were utilized to evaluate purity by SDS-PAGE [3,8,15].
Effect of amidine derivatives on NOS-promastigotes activity
The activity of NOS (in presence/absence of drug) purified fromLeishmaniaparasites was determined by measuring the decrease in the absorbance (JEWAY 6405 Spectrophotometer,U.K.) at 340 nm of NADPH consumed during the conversion of L- arginine to L-citrulline by NOS. The complete enzyme reaction mixture contained 50 mmol/L pot- assium phosphate buffer (pH 7.4), 1 mmol/L CaCl2, 0.1 mmol/L NADPH, 80mmol/L H4B, 10mmol/L FAD, 10mmol/L FMN, 0.1 mmol/LL-arginine, 2mg enzyme, methoxy-amidine ðLD50¼22mmol=LÞ or pentamidineðLD50¼0:46mmol=LÞin a final volume of 2 mL, at 258C. Blanks contained all the com- ponent’s, except L-arginine and the amidine com- pounds [8,22,32].
Data analysis
Three to five independent experiments were per- formed to quantify the levels of nitrite in supernatants cultures and determination of NOS activity. Data obtained with different treatments were analyzed statistically by 1-way ANOVA and Students t-test ðp,0:05Þ:
Results
Previous data from the authors laboratory has shown that the most effective amidine derivative against L. amazonensispromastigotes and axenic amastigotes was that with a methoxy group as substituent. In this work the effect of the amidine derivative on NO production of the parasite-macrophage interaction (24 h post infection) was evaluated. NO production when parasites were pre-treated with methoxy- amidine for 24 h before infection was higher (Figure 2a), compared with data observed when pentamidine was usedðp¼0:0194Þ; but the percen- tage of infection was flagrantly lower when the parasites were pre-treated with the methoxy com- pound (Figure 2b). The results show that after the infection have been installed, the methoxy derivative was able to destroy the interiorized parasites, without being hazardous to the host cell (Figure 2b). More- over, the methoxy derivative was also able to prevent macrophage infection with previously treated parasites (promastigotes incubated for 24 h/268C with the drug), even with a high production of nitrite (Figure 2a).
The presence of pentamidine did not have any effect in preventing infection under the same conditions
Figure 1. Chemical structure of Pentamidine Isethionate (a) and Methoxy-amidine (b).
since the parasites treated with the reference drug, were able to promote macrophage infection (100%) (Figure 2b).
Figure 3 showed that the methoxylated compound was able to inhibit significantly the purified NOS-L.
amazonensis promastigotes activity ðp¼0:0293Þ;
demonstrated by a lower consumption of the NADPH in the conversion ofL-arginine toL-citrulline.
Discussion
NO production appears to constitute one of the main microbiocidal mechanisms of murine macrophages and it has been implicated in the elimination of viruses, bacteria, fungi and protozoa [27]. Because of its variety of reaction partners (DNA, proteins,
low-molecular weight thiols, prosthetic groups, reactive oxygen intermediates), its widespread production by three different NOS and the fact that its activity is strongly influenced by its concentration, NO continues to surprise and perplex immunologists, parasitologists and biochemists researchers [4].
Resistance to leishmanial infections depends on killing the intracellular parasite by activated host macrophages through the L-arginine-NO metabolic pathway [26]. Recently, Holzmulleret al.(2002) [23]
have demonstrated that the exposure of amastigotes to moderate concentrations of NO-donating compounds (acidified sodium nitrite or nitrosylated albumin) or to endogenous NO produced by lipopolysaccharide or gamma interferon treatment of infected macrophages, resulted in a dramatic time-dependent cell death.
Figure 2. NO released (24 h post-infection) and (a) detected as nitrite in supernatant culture ofL. amazonensis-macrophage interaction, (b) analyzed comparatively with percentage of infection. Where M ¼macrophage culture, P¼ parasites and D¼drug. The protocol variables included: M¼ only macrophage culture; MþPþD ¼ macrophage infected withL. amazonensisand then treated with the drug;
M(Pþ D)¼L. amazonensisparasites were pre-treated with the drug for 24 h and then the macrophages were infected.
R.M. Temporalet al.
16
While other parasites can also infect macrophages, Leishmaniais distinctive in that not only does it rely on its own defenses to survive and reproduce within the macrophage phagolysosome, Leishmania also mani- pulates the host immune response in order to protect itself and to gain entry into the cell. These unique adaptive mechanisms help to promote survival of the Leishmania.
Studies performed on mice infected with L. major, have demonstrated that host defense against this infection depends on the interleukin-12-driven expan- sion (IL-12) of the T helper 1 cell subset, with production of cytokines, such as interferon-gamma, which activates the macrophages for killing the parasite through release of NO. The microbiocidal role of this radical is now emerging also in the human and canine model [5,27].
In this work, it was possible to observe that NO production (detected as nitrite in culture supernatant) during the host cell-parasite interaction is directly proportional to percentage of infection. Moreover, the most important data observed is the absence of infection when parasites (treated/non-treated) were added to macrophages (without drugs) in comparison to pentamidine isethionate. Under the same conditions this observation was detected at 24 h post infection, and additionally demonstrates that methoxy-amidine did not interfere with NO production by macrophages, as observed in the control. Analysis of the results analysis have shown that methoxy-amidine acts on the defense systems of L. amazonensis, corroborating the data obtained by Genestra and collaborators [19]. At 24 h, the higher detected nitrite level when the parasites were pre-incubated with methoxy-amidine can be due to
intense parasite killing post interaction parasite-macro- phage, maybe because the methoxy derivative interferes with the cross-talk of macrophage-parasites.
Previously, the authors analyzed the pathways signs in L. amazonensis promastigotes and the results showed a significant increase in PKG activity in the promastigotes culture containing a high percentage of metacyclic forms [13]. Therefore, the authors studied the existence of the NO/guanylate cyclase (GC)/cyclic guanosine monophosphate (GMP)/protein kinase G (PKG) pathway in these parasites, and they have associated the involvement of Leishmania-NOS enzyme systems in mechanisms of infectivity involving macrophages and also perhaps as a drug target. The dataobtained corroborate the view that this pathway is present in L. amazonensis, the etiological agent of various forms of the Leishmaniasis. In past analysis of NO production by these parasites in supernatant culture, the results revealed a significantly higher NO22 (a byproduct of NO) concentration in infective promastigotes, when compared with parasites with n passage (non infective promastigotes, without meta- cyclic forms detectable) [18,19].
In this work, a NOS was purified from the soluble extracts of L. amazonensispromastigotes forms. The purification protocol was similar to the employed for the purification of the L. donovani NOS-enzyme [3]
andT. cruziNOS-enzyme [30]. Previous results from the purification protocol have demonstrated that the representative silver-stained SDS gel shows that the purified preparation of L. amazonensis promas- tigotes NOS contained one single protein, which we believe to be aLeishmaniaNOS subunit [15,18].
Among the several amidine derivatives [12] tested by the authors group on Leishmania amazonensis, they defined as the most effective compound that with a methoxy group as substituent [6,7] (Figure 1). The drug showed a high effectiveness againstTrypanosoma evansi trypomastigotes, T. cruzi and PKA-L. amazo- nensis activity [6,14,20]. Following this data, the authors analyzed the effect of these compounds, in comparison with pentamidine (drug reference), on NO production by L. amazonensis promastigotes and axenic amastigotes by measuring nitrite, a by-product of NO released into the culture supernatants. The results indicated that NO production by infective promastigotes was significantly inhibited by methoxy- amidine [17].
Amidine derivatives contains a chemical structure shared by the guanidine group of the NOS substrate
L-arginine, suggesting the possibility of an interaction with this enzyme or electronic factors that will alter physical and chemical properties significant for NOS activity. Initially, the authors hypothesized that these drug can only inhibit the L-arginine transport of the Leishmaniaprotozoan parasite [25]. Then, in order to verify the effect of methoxy-amidine on purified NOS- L. amazonensis,they carried out an assay to analyze if
Figure 3. Activity of constitutive nitric oxide synthase (cNOS) of L- amazonensis promastigotes analyzed by the consumption of NADPH. When the enzyme was incubated with the methoxy derivativeðLD50¼14mMÞ; the consumption of the cofactor was lower, in comparison with when the enzyme was incubated with pentamidine (reference drug,ðLD50¼0:46mMÞ). This data shows that the NOS-promastigotes activity of L. amazonensis is significantlyðp¼0:0293Þaffected by methoxy-amidine.
whether there was a direct action of these compounds on NADPH consumption by this enzyme when the mixture reaction contained all the components involved in NO production. The results showed that the methoxylated compound was able to significantly inhibit the purified NOS-L. amazonensisactivityðp¼ 0:0293Þ; demonstrated by the low consumption of NADPH on conversion of L-arginine to L-citrulline.
This suggests that the methoxy-amidine interferes directly with NOS-Leishmania, through an unknown mechanism. Therefore, it seems that these mechan- isms of action are involved in the reduction of macrophage infection observed “in vitro”, corrobo- rated by the fact that the methoxy-derivative is non toxic and does not interfere with the morphology of the host cell [34].
In general, the electronic factors (s substituent), such as the inductive and the resonance effects are responsible for alterations in the physical and chemical properties of the compound, reflecting its biological activity. It is known that covalent bond formation between the drug and the target is not the only essential factor for a strict binding receptor-ligand; electrostatic interactions, hydrogen bonding and hydrophobic interactions can be equally important for the mechan- ism of action [2]. Based in thedatapresented here it is suggested that the NOS activity ofL. amazonensisis a possible target for amidine compounds.
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